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Protective Immunity to Streptococcus mutans Induced by Nasal Vaccination with Surface Protein Antigen and Mutant Cholera Toxin Adjuvant

  1. Masayuki Saito1,
  2. Shigeo Otake1,
  3. Mari Ohmura6,
  4. Masatomo Hirasawa2,
  5. Kazuko Takada2,
  6. Junichi Mega3,
  7. Ichiro Takahashi4,
  8. Hiroshi Kiyono4,
  9. Jerry R. McGhee6,
  10. Yoshifumi Takeda5 and
  11. Masafumi Yamamoto1,4
  1. Departments of
  2. 1Clinical Pathology,
  3. 2Microbiology, and
  4. 3Dentistry for the Handicapped, Nihon University School of Dentistry at Matsudo, Chiba,
  5. 4Department of Mucosal Immunology, Research Institute for Infectious Diseases, Osaka University, Osaka, and
  6. 5National Institute of Infectious Diseases, Tokyo, Japan;
  7. 6Department of Microbiology, Immunobiology Vaccine Center, University of Alabama at Birmingham
  1. Reprints or correspondence: Dr. Masafumi Yamamoto, Dept. of Clinical Pathology, Nihon University School of Dentistry at Matsudo, 2-870-1 Sakaecho-Nishi, Matsudo, Chiba 271-8587, Japan (fumi{at}mascat.nihon-u.ac.jp)
 
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Abstract

In this study, mice were immunized nasally with surface protein antigen of Streptococcus mutans serotype c (PAc) and a nontoxic A subunit mutant of cholera toxin (mCT) E112K, as a mucosal adjuvant. Immunization with PAc and mCT elicited significant PAc-specific secretory IgA in saliva and in nasal secretions. Antibody-forming cell (AFC) analysis confirmed the antibody (Ab) titers by revealing significant numbers of PAc-specific IgA AFCs in the submandibular gland and nasal passages. Furthermore, CD4+ T cells from cervical lymph nodes exhibited significant proliferative responses when restimulated with PAc in vitro. Importantly, mice that were nasally immunized with PAc plus mCT E112K exhibited significantly reduced oral colonization by S. mutans These results show that nasal administration of PAc and mCT E112K is potentially an effective mucosal vaccine against dental caries and reduces the colonization of S. mutans in the oral cavity

It is well known that Streptococcus mutans is the major pathogen of dental caries [1]. S. mutans produces a cell-surface protein antigen (Ag) with a molecular mass of 190 kDa, referred to as the surface protein antigen of S. mutans serotype c (PAc) [2], antigen I/II, protein B, or P1 [3]. Because PAc has been shown to be a key virulence factor for tooth adherence by S. mutans [3], blocking or inhibition of this cell surface adhesin by secretory IgA (S-IgA) in saliva is a logical approach for the prevention of initial colonization of teeth by S. mutans In this regard, it has been shown that nasal or oral immunization with PAc chemically conjugated with commercial preparations of B subunits of cholera toxin (CT-B), which contain trace amounts of the holotoxin, elicits significant Ag-specific salivary IgA antibody (Ab) responses [4, 5]. Furthermore, a fusion protein made with the saliva-binding region of PAc and CT-B induced high titers of anti–PAc salivary IgA Abs when given orally or nasally [6]. Thus, mucosally administered PAc plus CT appears to be an effective vaccine for induction of PAc-specific salivary IgA Ab responses; however, an enterotoxin adjuvant that causes clinical manifestations of cholera is unsuitable for use in humans. To eliminate the diarrheagenicity but retain the adjuvanticity of CT, we generated 2 mutants of CT (mCTs: S61F and E112K), which harbor single amino acid substitutions in the ADP-ribosyltransferase active center that render them enzymatically inactive and thus nontoxic; however, these mCTs still supported Ag-specific immune responses when administered nasally [7, 8]

In this study, we evaluated the efficacy of a mucosal vaccine, containing PAc plus mCT E112K, which was developed to prevent dental caries. Furthermore, to determine whether T cell–dependent Ab responses were generated in the mucosal compartment, PAc-specific proliferative responses of CD4+ T cells in cervical lymph nodes (CLNs) were examined

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Materials and Methods

MiceBALB/c mice were purchased from Charles River Japan and were maintained under pathogen-free conditions. All mice were provided with sterile food and water ad libitum and were used in this study at 8–12 weeks of age

Antigens and adjuvantsPAc from S. mutans MT8148 was purified by using a DEAE-sephacel (Amersham Pharmacia Biotech) and Sephacryl S200 (Amersham Pharmacia Biotech) from culture supernatants, as described elsewhere [9], with minor modifications. The mCT E112K was purified by using a d-galactose–immobilized column (Pierce) from a cell suspension prepared by sonication of the Escherichia coli containing the mCT E112K gene, as described elsewhere [7]. The purity of PAc and mCT E112K was assessed by SDS-PAGE, and no contaminating protein bands were noted

Immunization and sample collectionMice were immunized nasally on days 0, 7, and 14 with a 10-μL aliquot (5 μL per nostril) containing 10 μg of PAc alone or combined with 5 μg of mCT E112K. Saliva and nasal washes were collected, as described elsewhere [8], on day 21, to examine PAc-specific Ab responses

Detection of Ag-specific IgA Ab titers and Ab-forming cells (AFCs)IgA Ab titers in saliva and nasal washes were determined by ELISA, as described elsewhere [8]. End point titers were expressed as the reciprocal log2 of the last dilution that gave an optical density at 414 nm >0.1 units above those obtained with unimmunized mouse groups after 15 min of incubation. Enumeration of AFCs from the salivary gland and nasal passages was performed by an ELISPOT assay, as described elsewhere [8]

Stimulation of PAc-specific CD4+ T cellsCD4+ T cells from CLNs were purified by use of a magnet-activated cell sorter system (Miltenyi Biotec), as described elsewhere [8]. Purified CD4+ T cells (2×106 cells/mL) were cultured with T cell–depleted, mitomycin-treated splenic feeder cells (2.5×106 cells/mL) from unimmunized mice in the presence of 1 μg/mL of PAc in complete medium containing 10 U/mL of mouse recombinant interleukin-2 (Genzyme). The cultures were incubated for 4 days at 37°C in 5% CO2 in air. To measure PAc-specific CD4+ T cell proliferative responses, [3H] thymidine was added into the culture 18 h before termination

Oral colonization ofS. mutans. Streptomycin-resistant S. mutans MT8148R was kindly provided by Drs. S. Kawabata and S. Hamada (Osaka University). The oral colonization assay was carried out as described elsewhere, with minor modifications [5]. In brief, 2 weeks after the last immunization, the mice were given an ordinary diet (Sankyo Lab) containing tetracycline (4 mg/g of food) and deionized water containing penicillin G (4000 U/mL of water) for 4 days. The mice were then infected orally with 2×108 cfu of streptomycin-resistant S. mutans MT8148R on 10 consecutive days. Oral swabs were taken from all mice 2 weeks after the final challenge

Statistical analysisThe results are expressed as mean±SE, and statistical significance (P<.05) was determined by Student’s t test

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Results

Induction of PAc-specific Ab responses in mucosal compartmentsBecause a previous study [5] showed that PAc-specific salivary IgA Ab can reduce oral colonization by S. mutans it was important to assess IgA Ab responses in saliva. Nasal administration of PAc and mCT E112K induced PAc-specific IgA Ab titers in saliva (log2, 6.1±1.7) and in nasal wash samples (log2, 8.2±1.5). In contrast, essentially no Abs (<log2, 2) were detected in either saliva or nasal wash samples of mice that received nasal PAc or mCT E112K alone. AFC responses supported the Ab titers and showed that significant numbers of PAc-specific IgA-producing cells were detected in the submandibular gland and nasal passages following nasal administration of PAc plus mCT E112K. In contrast, few AFCs were found in the submandibular gland and nasal passages of mice given PAc alone (figure 1A)

Figure 1
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Figure 1

Characterization of surface protein antigen of Streptococcus mutans serotype c (PAc)–specific IgA antibody (A) and CD4+ T cell proliferation (B) in mucosal compartment of mice immunized nasally with PAc and a nontoxic A subunit mutant of cholera toxin (mCT E112K). Groups of BALB/c mice were immunized nasally with 100 ng of PAc plus 5 μg of mCT E112K (▪) or with 100 ng of PAc alone (□) on days 0, 7, and 14, and IgA antibody–forming cell (AFC) responses in the salivary gland and nasal passages were assessed on day 21. CD4+ T cells were isolated from cervical lymph nodes and were cultured with 1 μg/mL of PAc, in the presence of mitomycin-treated SP feeder cells as antigen-presenting cells and recombinant interleukin-2, for 4 days. The results are expressed as mean±SE values from 5 mice per group and were taken from a total of 3 separate experiments. *P<.05, compared with mice immunized with PAc alone

Ag-specific CD4+ T cell responsesTo determine whether Ag-specific mucosal IgA Abs induced by nasal immunization with PAc plus mCT E112K were T cell–dependent responses, we next examined PAc-specific CD4+ T cell proliferative responses. When CD4+ T cells isolated from CLNs of mice immunized with PAc plus mCT E112K were restimulated with PAc in vitro, significant levels of proliferative responses were induced. In contrast, essentially no increased proliferation occurred in CLNs taken from mice given PAc alone (figure 1B)

Nasally induced PAc-specific immunity suppresses colonization ofS. mutans.Since nasal PAc plus mCT E112K elicited Ag-specific S-IgA Ab responses, we sought to determine whether these Abs were capable of suppressing colonization of the tooth enamel by S. mutans Thus, mice given PAc plus mCT E112K, PAc alone, or mCT E112K alone were infected orally with streptomycin-resistant S. mutans MT8148R. Ag-specific immune responses induced by nasal immunization with PAc plus mCT E112K provided significant inhibition of colonization of S. mutans In contrast, colonization inhibition failed to occur in all mice given PBS, PAc, or mCT E112K alone (figure 2)

Figure 2
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Figure 2

Inhibition of oral colonization of Streptococcus mutans by nasal vaccine containing surface protein antigen of S. mutans serotype c (PAc) plus a nontoxic A subunit mutant of cholera toxin (mCT E112K). Groups of BALB/c mice were immunized nasally with PAc plus mCT E112K or with PAc alone, as described in the legend of figure 1. Nineteen days after the last immunization, the mice were infected orally with 2×108 cfu of streptomycin-resistant S. mutans MT8148R on 10 consecutive days. *P<.05, compared with mice immunized with PAc alone

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Discussion

In the present study, we have assessed the potential for application of a nontoxic mucosal adjuvant mCT E112K for development of a nasal vaccine for prevention of oral infection caused by S. mutans Our results showed that nasally administered PAc and a nontoxic A subunit mutant of CT E112K elicited PAc-specific S-IgA Ab responses in mucosal compartments, including the oral cavity. Furthermore, S-IgA anti-PAc Abs in saliva induced by nasal PAc plus mCT E112K inhibited the colonization of teeth by S. mutans

Although the demineralization of enamel results from acid production, adherence to and colonization of teeth by S. mutans are equally important for establishment of dental caries [10]. Tooth colonization by this oral bacteria appears to be a 2-step process, consisting of a sucrose-independent initial adherence to the acquired salivary pellicle, followed by sucrose-dependent cellular accumulation. In this regard, PAc has been shown to possess the function of cell-surface salivary adhesins and thus contributes to the success of oral streptococci in the colonization of the oral cavity [3]. In support of this, numerous studies have assessed the potential of PAc as a caries vaccine candidate and have shown that PAc is an effective Ag for induction of protective immune responses against S. mutans infection [3]. However, nasal immunization with PAc alone failed to enhance anti-PAc Abs in mucosal compartments. Indeed, other studies also reported that PAc without an appropriate adjuvant is a weak immunogen when given via the mucosal route [4]. These studies, together with our present results, indicate that a mucosal adjuvant such as CT is required for induction of PAc-specific Ab responses in mucosal compartments when PAc is given by mucosal routes such as the nasal one

CT has been widely used as an effective adjuvant in studies of anti-caries vaccine [3–5]. However, despite these beneficial attributes of CT, the native form of the toxin is unsuitable for use in humans, because the A subunit of CT (CT-A) catalyzes ADP-ribosylation of Gsα, resulting in elevated intracellular cyclic AMP levels, which, in epithelial cells, cause secretion of water and chloride ions into the small intestine, leading to the development of a watery diarrhea [11]. Although recent studies demonstrated that recombinant CT-B contributes some adjuvant properties to PAc when given nasally [12], it is likely that CT-B acts mainly as a targeting protein for GM1 ganglioside, and toxicity-associated CT-A is required for development of full adjuvanticity [7, 13]. However, the ADP-ribosyltransferase activity of CT-A must be eliminated before it can be used to develop a caries vaccine for humans. In this regard, several groups also reported that single amino acid–substituted mutants of heat-labile toxin of E. coli (e.g., R7K, S63K, and R192G), which lack ADP-ribosyltransferase activity, were shown to retain some adjuvant properties [14–16]. The studies presented here also demonstrate that mCT E112K, which lacks ADP-ribosyltransferase activity, is an effective mucosal adjuvant for nasal immunization, and that, when given with PAc, it facilitates the development of protective immunity against oral colonization of S. mutans These findings suggest that mCT E112K is an attractive candidate as a mucosal adjuvant for the development of a caries vaccine

The mechanisms by which mCT E112K acts as an effective mucosal adjuvant for induction and regulation of S-IgA Ab responses in mucosal tissues have not been elucidated. In this regard, several studies have reported that costimulatory signals, via B7-2, play a critical role in adjuvanticity induced by CT [17]. Thus, CT up-regulated B7-2 on bone marrow–derived macrophages, and, when treated with CT, the macrophages enhanced T cell proliferative responses [17]. In those studies, it was concluded that enhancement of B7 expression on APCs by CT was dependent on ADP-ribosyltransferase activity. However, our recent studies have shown that mCT E112K, like native CT, enhances B7-2 expression on Peyer’s patch B cells and macrophages, with subsequent enhancement of T cell proliferative responses in the presence of anti-CD3 stimulation [18]. Thus, CT induces adjuvant responses, in part, via up-regulation of B7-2 on APCs, which is independent of ADP-ribosyltransferase activity

In summary, our study has demonstrated that nasal vaccine containing S. mutans PAc plus mCT E112K elicited PAc-specific S-IgA Ab responses in mucosal compartments, including the oral cavity. Furthermore, these PAc-specific Ab responses inhibited colonization of tooth enamel surfaces by S. mutans The combination of PAc and mCT E112K could be considered to be a new form of nasal vaccine for induction of salivary IgA to PAc, and thus it may be applied as a practical regimen for vaccinating humans against dental caries

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Acknowledgments

Shigetada Kawabata and Shigeyuki Hamada (Osaka University) kindly provided streptomycin-resistant Streptococcus mutans MT8148R

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Footnotes

  • All experiments described herein were approved by the component local authorities. All procedures were done in agreement with National Institutes of Health guidelines for the handling of laboratory animals

    Financial support: US Public Health Service grants AI18958, DK44240, and AI43197 and contracts AI65298 and AI65299; Grant-in-Aid for Encouragement of Young Scientists (11771149) from Japan Society for the Promotion of Science; grant from Research for the Frontier and Science; and Grant-in-Aid for Center of Excellence Research (09CE2007) from the Japanese Ministry of Education, Science, Sports, and Culture

  • Received July 31, 2000.
  • Revision received November 21, 2000.
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  1. J Infect Dis. (2001) 183 (5): 823-826. doi: 10.1086/318826
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